Substrate recognition by class I lysyl-tRNA synthetases: a molecular basis for gene displacement

Abstract

Lysyl-tRNA synthetases (LysRSs) are unique amongst the aminoacyl-tRNA synthetases in being composed of unrelated class I and class II enzymes. To allow direct comparison between the two types of LysRS, substrate recognition by class I LysRSs was examined. Genes encoding both an archaeal and a bacterial class I enzyme were able to rescue an Escherichia coli strain deficient in LysRS, indicating their ability to functionally substitute for a class II LysRS in vivo. In vitro characterization showed lysine activation and recognition to be tRNA-dependent, an attribute of several class I, but not class II, aminoacyl-tRNA synthetases. Examination of tRNA recognition showed that class I LysRSs recognize the same elements in tRNALys as their class II counterparts, namely the discriminator base (N73) and the anticodon. This sequence-specific recognition of the same nucleotides in tRNALys by the two unrelated types of enzyme suggests that tRNALys predates at least one of the LysRSs in the evolution of the translational apparatus. The only observed variation in recognition was that the G2.U71 wobble pair of spirochete tRNALys acts as antideterminant for class II LysRS but does not alter class I enzyme recognition. This difference in tRNA recognition strongly favors the use of a class I-type enzyme to aminoacylate particular tRNALys species and provides a molecular basis for the observed displacement of class II by class I LysRSs in certain bacteria.

Figure 1

Unrooted phylogeny of class I LysRSs. Phylogenies were constructed by using the maximum likelihood method (1,000 puzzling steps) implemented in the program puzzle 4.0 (48) and protein parsimony methods (200 bootstrap replicates) implemented in the Phylip package version 3.5c (J. Felsenstein, University of Washington). Numbers represent the percentage occurrence of nodes from maximum likelihood and protein parsimony methods, respectively. Sequences were aligned by using the program clustal x (49).

Figure 2

Predicted secondary structure of unmodified tRNA^Lys from B. burgdorferi, E. coli, and M. maripaludis. The anticodon and discriminator base are circled; the G2⋅U71 wobble pair is boxed.

Figure 3

(A) Pyrophosphate exchange by M. maripaludis LysRS. The reaction was incubated for 20 min at 37°C in the absence of various components as indicated. Values represent micromoles of ATP in a 20-μl aliquot. (B) In vitro Ap₄A biosynthesis. Reactions were performed in the presence of E. coli cell-free extract (•), M. maripaludis LysRS (○), or B. burgdorferi LysRS (■). Values represent picomoles of Ap₄A in a 50-μl aliquot.